Process for Preparing L-amino Acids

ABSTRACT

The invention relates to isolated polynucleotides comprising a polynucleotide sequence chosen from the group consisting of a) polynucleotide which is identical to the extent of at least 70% to a polynucleotide which codes for a polypeptide which comprises the amino acid sequence of SEQ ID No. 2, b) polynucleotide which codes for a polypeptide which comprises an amino acid sequence which is identical to the extent of at least 70% to the amino acid sequence of SEQ ID No. 2, c) polynucleotide which is complementary to the polynucleotides of a) or b), and d) polynucleotide comprising at least 15 successive nucleotides of the polynucleotide sequence of a), b) or c), and a process for the fermentative preparation of L-amino acids using coryneform bacteria in which at least the citA gene is present in attenuated form, and the use of polynucleotides which comprise the sequences according to the invention as hybridization probes.

The invention provides nucleotide sequences from coryneform bacteriawhich code for the citA gene and a process for the fermentativepreparation of amino acids, in particular L-lysine, by attenuation ofthe citA gene. The citA gene codes for the sensor kinase CitA of atwo-component system.

PRIOR ART

L-Amino acids, in particular L-lysine, are used in human medicine and inthe pharmaceuticals industry, in the foodstuffs industry and veryparticularly in animal nutrition.

It is known that amino acids are prepared by fermentation from strainsof coryneform bacteria, in particular Corynebacterium glutamicum.Because of their great importance, work is constantly being undertakento improve the preparation processes. Improvements to the process canrelate to fermentation measures, such as, for example, stirring andsupply of oxygen, or the composition of the nutrient media, such as, forexample, the sugar concentration during the fermentation, or the workingup to the product form by, for example, ion exchange chromatography, orthe intrinsic output properties of the microorganism itself.

Methods of mutagenesis, selection and mutant selection are used toimprove the output properties of these microorganisms. Strains which areresistant to antimetabolites or are auxotrophic for metabolites ofregulatory importance and which produce amino acids are obtained in thismanner.

Methods of the recombinant DNA technique have also been employed forsome years for improving the strain of Corynebacterium strains whichproduce L-amino acids.

OBJECT OF THE INVENTION

The inventors had the object of providing new measures for improvedfermentative preparation of amino acids, in particular L-lysine.

DESCRIPTION OF THE INVENTION

If L-lysine or lysine are mentioned in the following, this also meansthe salts, such as e.g. lysine monohydrochloride or lysine sulfate.

The invention provides an isolated polynucleotide from coryneformbacteria, comprising a polynucleotide sequence which codes for the citAgene chosen from the group consisting of

-   a) polynucleotide which is identical to the extent of at least 70%    to a polynucleotide which codes for a polypeptide which comprises    the amino acid sequence of SEQ ID No. 2,-   b) polynucleotide which codes for a polypeptide which comprises an    amino acid sequence which is identical to the extent of at least 70%    to the amino acid sequence of SEQ ID No. 2,-   c) polynucleotide which is complementary to the polynucleotides    of a) or b), and-   d) polynucleotide comprising at least 15 successive nucleotides of    the polynucleotide sequence of a), b) or c),    the polypeptide preferably having the activity of sensor kinase    CitA.

The invention also provides the abovementioned polynucleotide, thispreferably being a DNA which is capable of replication, comprising:

-   (i) the nucleotide sequence shown in SEQ ID No.1 or-   (ii) at least one sequence which corresponds to sequence (i) within    the range of the degeneration of the genetic code, or-   (iii) at least one sequence which hybridizes with the sequences    complementary to sequences (i) or (ii), and optionally-   (iv) sense mutations of neutral function in (i).

The invention also provides:

-   a polynucleotide, in particular DNA, which is capable of replication    and comprises the nucleotide sequence as shown in SEQ ID No.1;-   a polynucleotide which codes for a polypeptide which comprises the    amino acid sequence as shown in SEQ ID No. 2;-   a vector containing parts of the polynucleotide according to the    invention, but at least 15 successive nucleotides of the sequence    claimed-   and coryneform bacteria in which the citA gene is attenuated, in    particular by an insertion or deletion.

The invention also provides polynucleotides which substantially comprisea polynucleotide sequence, which are obtainable by screening by means ofhybridization of a corresponding gene library of a coryneform bacterium,which comprises the complete gene or parts thereof, with a probe whichcomprises the sequence of the polynucleotide according to the inventionor a fragment thereof, and isolation of the polynucleotide sequencementioned.

Polynucleotides comprising the sequences according to the invention aresuitable as hybridization probes for RNA, cDNA and DNA, in order toisolate, in the full length, nucleic acids or polynucleotides or geneswhich code for CitA protein or to isolate those nucleic acids orpolynucleotides or genes which have a high similarity with the sequenceof the citA gene.

Polynucleotides comprising the sequences according to the invention arefurthermore suitable as primers with the aid of which DNA of genes whichcode for the CitA protein can be prepared by the polymerase chainreaction (PCR).

Such oligonucleotides which serve as probes or primers comprise at least30, preferably at least 20, very particularly preferably at least 15successive nucleotides. Oligonucleotides which have a length of at least40 or 50 nucleotides are also suitable.

“Isolated” means separated out of its natural environment.

“Polynucleotide” in general relates to polyribonucleotides andpolydeoxyribonucleotides, it being possible for these to be non-modifiedRNA or DNA or modified RNA or DNA.

The polynucleotides according to the invention include a polynucleotideaccording to SEQ ID No. 1 or a fragment prepared therefrom and alsothose which are at least 70%, preferably at least 80% and in particularat least 90% to 95% identical to the polynucleotide according to SEQ IDNo. 1 or a fragment prepared therefrom.

“Polypeptides” are understood as meaning peptides or proteins whichcomprise two or more amino acids bonded via peptide bonds.

The polypeptides according to the invention include a polypeptideaccording to SEQ ID No. 2, in particular those with the biologicalactivity of the CitA protein and also those which are at least 70%,preferably at least 80% and in particular at least 90% to 95% identicalto the polypeptide according to SEQ ID No. 2 and have the activitymentioned.

The invention moreover relates to a process for the fermentativepreparation of amino acids, in particular L-lysine, using coryneformbacteria which in particular already produce amino acids and in whichthe nucleotide sequences which code for the citA gene are attenuated, inparticular eliminated or expressed at a low level.

The term “attenuation” in this connection describes the reduction orelimination of the intracellular activity of one or more enzymes(proteins) in a microorganism which are coded by the corresponding DNA,for example by using a weak promoter or using a gene or allele whichcodes for a corresponding enzyme with a low activity or inactivates thecorresponding gene or enzyme (protein), and optionally combining thesemeasures.

The microorganisms which the present invention provides can prepareamino acids, in particular L-lysine, from glucose, sucrose, lactose,fructose, maltose, molasses, starch, cellulose or from glycerol andethanol. They can be representatives of coryneform bacteria, inparticular of the genus Corynebacterium. Of the genus Corynebacterium,there may be mentioned in particular the species Corynebacteriumglutamicum, which is known among experts for its ability to produceL-amino acids.

Suitable strains of the genus Corynebacterium, in particular of thespecies Corynebacterium glutamicum (C. glutamicum), are in particularthe known wild-type strains

Corynebacterium glutamicum ATCC13032

Corynebacterium acetoglutamicum ATCC15806

Corynebacterium acetoacidophilum ATCC13870

Corynebacterium melassecola ATCC17965

Corynebacterium thermoaminogenes FERM BP-1539

Brevibacterium flavum ATCC14067

Brevibacterium lactofermentum ATCC13869 and

Brevibacterium divaricatum ATCC14020

or L-amino acid-producing mutants or strains prepared therefrom, suchas, for example, the L-lysine-producing strains

Corynebacterium glutamicum FERM-P 1709

Brevibacterium flavum FERM-P 1708

Brevibacterium lactofermentum FERM-P 1712

Corynebacterium glutamicum FERM-P 6463

Corynebacterium glutamicum FERM-P 6464

Corynebacterium glutamicum DM58-1

Corynebacterium glutamicum DG52-5

Corynebacterium glutamicum DSM 5715 and

Corynebacterium glutamicum DSM 12866

The inventors have succeeded in isolating the new citA gene of C.glutamicum which codes for the CitA protein and which is a sensor kinaseof a two-component system.

To isolate the citA gene or also other genes of C. glutamicum, a genelibrary of this microorganism is first set up in Escherichia coli (E.coli). The setting up of gene libraries is described in generally knowntextbooks and handbooks. The textbook by Winnacker: Gene und Klone, EineEinführung in die Gentechnologie [Genes and Clones, An Introduction toGenetic Engineering] (Verlag Chemie, Weinheim, Germany, 1990), or thehandbook by Sambrook et al.: Molecular Cloning, A Laboratory Manual(Cold Spring Harbor Laboratory Press, 1989) may be mentioned as anexample. A well-known gene library is that of the E. coli K-12 strainW3110 set up in λ-vectors by Kohara et al. (Cell 50, 495-508 (1987)).Bathe et al. (Molecular and General Genetics, 252:255-265, 1996)describe a gene library of C. glutamicum ATCC13032, which was set upwith the aid of the cosmid vector SuperCos I (Wahl et al., 1987,Proceedings of the National Academy of Sciences USA, 84:2160-2164) inthe E. coli K-12 strain NM554 (Raleigh et al., 1988, Nucleic AcidsResearch 16:1563-1575). Börmann et al. (Molecular Microbiology 6(3),317-326 (1992)) in turn describe a gene library of C. glutamicumATCC13032 using the cosmid pHC79 (Hohn and Collins, 1980, Gene 11,291-298).

To prepare a gene library of C. glutamicum in E. coli it is alsopossible to use plasmids such as pBR322 (Bolivar, 1979, Life Sciences,25, 807-818) or pUC9 (Vieira et al., 1982, Gene, 19:259-268). Suitablehosts are, in particular, those E. coli strains which are restriction-and recombination-defective, such as, for example, the strain DH5α(Jeffrey H. Miller: “A Short Course in Bacterial Genetics, A LaboratoryManual and Handbook for Escherichia coli and Related Bacteria”, ColdSpring Harbour [sic] Laboratory Press, 1992).

The long DNA fragments cloned with the aid of cosmids or other λ-vectorscan than be subcloned in turn into the usual vectors suitable for DNAsequencing.

Methods of DNA sequencing are described, inter alia, by Sanger et al.(Proceedings of the National Academy of Sciences of the United States ofAmerica USA, 74:5463-5467, 1977).

The resulting DNA sequences can then be investigated with knownalgorithms or sequence analysis programs, such as e.g. that of Staden(Nucleic Acids Research 14, 217-232(1986)), that of Marck (Nucleic AcidsResearch 16, 1829-1836 (1988)) or the GCG program of Butler (Methods ofBiochemical Analysis 39, 74-97 (1998)).

The new DNA sequence of C. glutamicum which codes for the citA gene andwhich, as SEQ ID No. 1, is a constituent of the present invention hasbeen found in this way. The amino acid sequence of the correspondingprotein has furthermore been derived from the present DNA sequence bythe methods described above. The resulting amino acid sequence of thecitA gene product is shown in SEQ ID No. 2.

Coding DNA sequences which result from SEQ ID No. 1 by the degeneracy ofthe genetic code are also a constituent of the invention. In the sameway, DNA sequences which hybridize with SEQ ID No. 1 or parts of SEQ IDNo. 1 are a constituent of the invention. Conservative amino acidexchanges, such as e.g. exchange of glycine for alanine or of asparticacid for glutamic acid in proteins, are furthermore known among expertsas “sense mutations” which do not lead to a fundamental change in theactivity of the protein, i.e. are of neutral function. It is furthermoreknown that changes on the N and/or C terminus of a protein cannotsubstantially impair or can even stabilize the function thereof.Information in this context can be found by the expert, inter alia, inBen-Bassat et al. (Journal of Bacteriology 169:751-757 (1987)), inO'Regan et al. (Gene 77:237-251 (1989)), in Sahin-Toth et al. (ProteinSciences 3:240-247 (1994)), in Hochuli et al. (Bio/Technology6:1321-1325 (1988)) and in known textbooks of genetics and molecularbiology. Amino acid sequences which result in a corresponding mannerfrom SEQ ID No. 2 are also a constituent of the invention.

Finally, DNA sequences which are prepared by the polymerase chainreaction (PCR) using primers which result from SEQ ID No. 1 are aconstituent of the invention. Such oligonucleotides typically have alength of at least 15 nucleotides.

Instructions for identifying DNA sequences by means of hybridization canbe found by the expert, inter alia, in the handbook “The DIG SystemUsers Guide for Filter Hybridization” from Boehringer Mannheim GmbH(Mannheim, Germany, 1993) and in Liebl et al. (International Journal ofSystematic Bacteriology 41: 255-260 (1991)). The hybridization takesplace under stringent conditions, that is to say only hybrids in whichthe probe and target sequence, i.e. the polynucleotides treated with theprobe, are at least 70% identical are formed. It is known that thestringency of the hybridization, including the washing steps, isinfluenced or determined by varying the buffer composition, thetemperature and the salt concentration. The hybridization reaction ispreferably carried out under a relatively low stringency compared withthe washing steps (Hybaid Hybridisation Guide, Hybaid Limited,Teddington, UK, 1996). A 5×SSC buffer at a temperature of approx. 50-68°C., for example, can be employed for the hybridization reaction. Probescan also hybridize here with polynucleotides which are less than 70%identical to the sequence of the probe. Such hybrids are less stable andare removed by washing under stringent conditions. This can be achieved,for example, by lowering the salt concentration to 2×SSC and optionallysubsequently 0.5×SSC (The DIG System User's Guide for FilterHybridisation, Boehringer Mannheim, Mannheim, Germany, 1995) atemperature of approx. 50-68° C. being established. Polynucleotidefragments which are, for example, at least 70% or at least 80% or atleast 90% to 95% identical to the sequence of the probe employed can beisolated by increasing the hybridization temperature stepwise from 50 to68° C. in steps of approx. 1-2° C. Further instructions on hybridizationare obtainable on the market in the form of so-called kits (e.g. DIGEasy Hyb from Roche Diagnostics GmbH, Mannheim, Germany, Catalogue No.1603558).

Instructions for amplification of DNA sequences with the aid of thepolymerase chain reaction (PCR) can be found by the expert, inter alia,in the handbook by Gait: OligonukleotidesA: [sic] a Practical Approach(IRL Press, Oxford, UK, 1984) and in Newton and Graham: PCR (SpektrumAkademischer Verlag, Heidelberg, Germany, 1994).

In the work on the present invention, it has been found that coryneformbacteria produce amino acids, in particular L-lysine, in an improvedmanner after attenuation of the citA gene.

To achieve an attenuation, either the expression of the citA gene or thecatalytic properties of the enzyme protein can be reduced or eliminated.The two measures can optionally be combined.

The reduction in gene expression can take place by suitable culturing orby genetic modification (mutation) of the signal structures of geneexpression. Signal structures of gene expression are, for example,repressor genes, activator genes, operators, promoters, attenuators,ribosome binding sites, the start codon and terminators. The expert canfind information on this e.g. in the patent application WO 96/15246, inBoyd and Murphy (Journal of Bacteriology 170: 5949 (1988)), in Voskuiland Chambliss (Nucleic Acids Research 26: 3548 (1998), in Jensen andHammer (Biotechnology and Bioengineering 58: 191 (1998)), in Pátek etal. (Microbiology 142: 1297 (1996)), Vasicova et al. (Journal ofBacteriology 181: 6188 (1999)) and in known textbooks of genetics andmolecular biology, such as e.g. the textbook by Knippers (“MolekulareGenetik [Molecular Genetics]”, 6th edition, Georg Thieme Verlag,Stuttgart, Germany, 1995) or that by Winnacker (“Gene und Klone [Genesand Clones]”, VCH Verlagsgesellschaft, Weinheim, Germany, 1990).

Mutations which lead to a change or reduction in the catalyticproperties of enzyme proteins are known from the prior art; exampleswhich may be mentioned are the works by Qiu and Goodman (Journal ofBiological Chemistry 272: 8611-8617 (1997)), Sugimoto et al. (BioscienceBiotechnology and Biochemistry 61: 1760-1762 (1997)) and Möckel (“DieThreonindehydratase aus Corynebacterium glutamicum: Aufhebung derallosterischen Regulation und Struktur des Enzyms [Threonine dehydratasefrom Corynebacterium glutamicum: Cancelling the allosteric regulationand structure of the enzyme]”, Reports from the Jülich, Research Centre,Jül-2906, ISSN09442952, Jülich, Germany, 1994). Summarizing descriptionscan be found in known textbooks of genetics and molecular biology, suchas e.g. that by Hagemann (“Allgemeine Genetik [General Genetics]”,Gustav Fischer Verlag, Stuttgart, 1986).

Possible mutations are transitions, transversions, insertions anddeletions. Depending on the effect of the amino acid exchange on theenzyme activity, missense mutations or nonsense mutations are referredto. Insertions or deletions of at least one base pair (bp) in a genelead to frame shift mutations, as a consequence of which incorrect aminoacids are incorporated or translation is interrupted prematurely.Deletions of several codons typically lead to a complete loss of theenzyme activity. Instructions on generation of such mutations are priorart and can be found in known textbooks of genetics and molecularbiology, such as e.g. the textbook by Knippers (“Molekulare Genetik[Molecular Genetics]”, 6th edition, Georg Thieme Verlag, Stuttgart,Germany, 1995), that by Winnacker (“Gene und Klone [Genes and Clones]”,VCH Verlagsgesellschaft, Weinheim, Germany, 1990) or that by Hagemann(“Allgemeine Genetik [General Genetics]”, Gustav Fischer Verlag,Stuttgart, 1986).

A common method of mutating genes of C. glutamicum is the method of genedisruption and gene replacement described by Schwarzer and Pühler(Bio/Technology 9, 84-87 (1991)).

In the method of gene disruption a central part of the coding region ofthe gene of interest is cloned in a plasmid vector which can replicatein a host (typically E. coli), but not in C. glutamicum. Possiblevectors are, for example, pSUP301 (Simon et al., Bio/Technology 1,784-791 (1983)), pK18mob or pK19mob (Schäfer et al., Gene 145, 69-73(1994)), pK18mobsacB or pK19mobsacB (Jäger et al., Journal ofBacteriology 174: 5462-65 (1992)), pGEM-T (Promega corporation, Madison,Wis., USA), pCR2.1-TOPO (Shuman (1994). Journal of Biological Chemistry269:32678-84; U.S. Pat. No. 5,487,993), pCR®Blunt (Invitrogen,Groningen, Holland; Bernard et al., Journal of Molecular Biology, 234:534-541 (1993)) or pEM1 (Schrumpf et al, 1991, Journal of Bacteriology173:4510-4516). The plasmid vector which contains the central part ofthe coding region of the gene is then transferred into the desiredstrain of C. glutamicum by conjugation or transformation. The method ofconjugation is described, for example, by Schäfer et al. (Applied andEnvironmental Microbiology 60, 756-759 (1994)). Methods fortransformation are described, for example, by Thierbach et al. (AppliedMicrobiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan(Bio/Technology 7, 1067-1070 (1989)) and Tauch et al. (FEMSMicrobiological Letters 123, 343-347 (1994)). After homologousrecombination by means of a “cross-over” event, the coding region of thegene in question is interrupted by the vector sequence and twoincomplete alleles are obtained, one lacking the 3′ end and one lackingthe 5′ end. This method has been used, for example, by Fitzpatrick etal. (Applied Microbiology and Biotechnology 42, 575-580 (1994)) toeliminate the recA gene of C. glutamicum.

In the method of gene replacement, a mutation, such as e.g. a deletion,insertion or base exchange, is established in vitro in the gene ofinterest. The allele prepared is in turn cloned in a vector which is notreplicative for C. glutamicum and this is then transferred into thedesired host of C. glutamicum by transformation or conjugation. Afterhomologous recombination by means of a first “cross-over” event whicheffects integration and a suitable second “cross-over” event whicheffects excision in the target gene or in the target sequence, theincorporation of the mutation or of the allele is achieved. This methodwas used, for example, by Peters-Wendisch et al. (Microbiology 144,915-927 (1998)) to eliminate the pyc gene of C. glutamicum by adeletion.

A deletion, insertion or a base exchange can be incorporated into thecitA gene in this manner.

In addition, it may be advantageous for the production of L-amino acids,in particular L-lysine, to enhance, in particular to over-express, oneor more enzymes of the particular biosynthesis pathway, of glycolysis,of anaplerosis, of the pentose phosphate cycle or of amino acid exportand optionally regulatory proteins, in addition to attenuation of thecitA gene.

The term “enhancement” in this connection describes the increase in theintracellular activity of one or more enzymes (proteins) in amicroorganism which are coded by the corresponding DNA, for example byincreasing the number of copies of the gene or genes, using a potentpromoter or using a gene or allele which codes for a correspondingenzyme (protein) having a high activity, and optionally combining thesemeasures.

Thus, for example, for the preparation of L-lysine, at the same time oneor more of the genes chosen from the group consisting of

-   the dapA gene which codes for dihydrodipicolinate synthase (EP-B 0    197 335),-   the gap gene which codes for glyceraldehyde 3-phosphate    dehydrogenase (Eikmanns (1992), Journal of Bacteriology    174:6076-6086),-   the zwf gene which codes for glucose 6-phosphate dehydrogenase    (JP-A-09224661),-   the pyc gene which codes for pyruvate carboxylase (DE-A-198 31 609),-   the lysE gene which codes for lysine export (DE-A-195-48 222)-   the lysC gene which codes for a feed back resistant aspartate kinase    (EP-B-0387527; EP-A-0699759; WO 00/63388), or-   the zwal gene which codes for the Zwal protein (DE: 199 59 328.0,    DSM 13115)    can be enhanced, in particular over-expressed.

It may furthermore be advantageous for the production of amino acids, inparticular L-lysine, in addition to the attenuation of the citA gene, atthe same time for one or more of the genes chosen from the groupconsisting of

-   the pck gene which codes for phosphoenol pyruvate carboxykinase (DE    199 50 409.1, DSM 13047),-   the pgi gene which codes for glucose 6-phosphate isomerase (U.S.    application Ser. No. 09/396,478, DSM 12969),-   the poxB gene which codes for pyruvate oxidase (DE:1995 1975.7, DSM    13114)-   the zwa2 gene which codes for the Zwa2 protein (DE: 199 59,327.2,    DSM 13113)    to be attenuated.

In addition to attenuation of the citA gene it may furthermore beadvantageous, for the production of amino acids, in particular L-lysine,to eliminate undesirable side reactions, (Nakayama: “Breeding of AminoAcid Producing Microorganisms”, in: Overproduction of MicrobialProducts, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK,1982).

The invention also provides the microorganisms prepared according to theinvention, and these can be cultured continuously or discontinuously inthe batch process (batch culture) or in the fed batch (feed process) orrepeated fed batch process (repetitive feed process) for the purpose ofproduction of L-amino acids, in particular L-lysine. A summary of knownculture methods is described in the textbook by Chmiel(Bioprozesstechnik 1. Einführung in die Bioverfahrenstechnik (BioprocessTechnology 1. Introduction to Bioprocess Technology (Gustav FischerVerlag, Stuttgart, 1991)) or in the textbook by Storhas [Bioreaktorenund periphere Einrichtungen (Bioreactors and Peripheral Equipment](Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).

The culture medium to be used must meet the requirements of theparticular strains in a suitable manner. Descriptions of culture mediafor various microorganisms are contained in the handbook “Manual ofMethods for General Bacteriology” of the American Society forBacteriology (Washington D.C., USA, 1981). Sugars and carbohydrates,such as e.g. glucose, sucrose, lactose, fructose, maltose, molasses,starch and cellulose, oils and fats, such as, for example, soya oil,sunflower oil, groundnut oil and coconut fat, fatty acids, such as, forexample, palmitic acid, stearic acid and linoleic acid, alcohols, suchas, for example, glycerol and ethanol, and organic acids, such as, forexample, acetic acid, can be used as the source of carbon. Thesesubstances can be used individually or as a mixture.

Organic nitrogen-containing compounds, such as peptones, yeast extract,meat extract, malt extract, corn steep liquor, soya bean flour and urea,or inorganic compounds, such as ammonium sulfate, ammonium chloride,ammonium phosphate, ammonium carbonate and ammonium nitrate, can be usedas the source of nitrogen. The sources of nitrogen can be usedindividually or as a mixture.

Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts can be used asthe source of phosphorus. The culture medium must furthermore comprisesalts of metals, such as, for example, magnesium sulfate or ironsulfate, which are necessary for growth. Finally, essential growthsubstances, such as amino acids and vitamins, can be employed inaddition to the abovementioned substances. Suitable precursors canmoreover be added to the culture medium. The starting substancesmentioned can be added to the culture in the form of a single batch, orcan be fed in during the culture in a suitable manner.

Basic compounds, such as sodium hydroxide, potassium hydroxide, ammoniaor aqueous ammonia, or acid compounds, such as phosphoric acid orsulfuric acid, can be employed in a suitable manner to control the pH ofthe culture. Antifoams, such as, for example, fatty acid polyglycolesters, can be employed to control the development of foam. Suitablesubstances having a selective action, such as, for example, antibiotics,can be added to the medium to maintain the stability of plasmids. Tomaintain aerobic conditions, oxygen or oxygen-containing gas mixtures,such as, for example, air, are introduced into the culture. Thetemperature of the culture is usually 20° C. to 45° C., and preferably25° C. to 40° C. Culturing is continued until a maximum of the desiredproduct has formed. This target is usually reached within 10 hours to160 hours.

Methods for the determination of L-amino acids are known from the priorart. The analysis can thus be carried out, for example, as described bySpackman et al. (Analytical Chemistry, 30, (1958), 1190) by anionexchange chromatography with subsequent ninhydrin derivatization, or itcan be carried out by reversed phase HPLC, for example as described byLindroth et al. (Analytical Chemistry (1979) 51: 1167-1174).

The invention furthermore relates to a process for the fermentativepreparation of an amino acid chosen from the group consisting ofL-asparagine, L-threonine, L-serine, L-glutamate, L-glycine, L-alanine,L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine,L-phenylalanine, L-histidine, L-lysine, L-tryptophan and L-arginine, inparticular L-lysine, using coryneform bacteria which in particularalready produce one or more of the amino acids mentioned.

The following microorganism was deposited on 23.01.2001 as a pureculture at the Deutsche Sammlung für Mikrorganismen [sic] undZellkulturen (DSMZ=German Collection of Microorganisms and CellCultures, Braunschweig, Germany) in accordance with the Budapest Treaty:

-   Escherichia coli strain Top10/pCR2.1citAint as DSM 13998.

The present invention is explained in more detail in the following withthe aid of embodiment examples.

The isolation of plasmid DNA from Escherichia coli and all techniques ofrestriction, Klenow and alkaline phosphatase treatment were carried outby the method of Sambrook et al. (Molecular Cloning. A LaboratoryManual, 1989, Cold Spring Harbour [sic] Laboratory Press, Cold SpringHarbor, N.Y., USA). Methods for transformation of Escherichia coli arealso described in this handbook.

The composition of the usual nutrient media, such as LB or TY medium,can also be found in the handbook by Sambrook et al.

EXAMPLES Example 1

Preparation of a Genomic Cosmid Gene Library from C. glutamicum ATCC13032

Chromosomal DNA from C. glutamicum ATCC 13032 was isolated as describedby Tauch et al. (1995, Plasmid 33:168-179) and partly cleaved with therestriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany,Product Description Sau3AI, Code no. 27-0913-02). The DNA fragments weredephosphorylated with shrimp alkaline phosphatase (Roche MolecularBiochemicals, Mannheim, Germany, Product Description SAP, Code no.1758250). The DNA of the cosmid vector SuperCos1 (Wahl et al. (1987),Proceedings of the National Academy of Sciences, USA 84:2160-2164),obtained from Stratagene (La Jolla, USA, Product Description SuperCos1Cosmid Vektor Kit, Code no. 251301) was cleaved with the restrictionenzyme XbaI (Amersham Pharmacia, Freiburg, Germany, Product DescriptionXbaI, Code no. 27-0948-02) and likewise dephosphorylated with shrimpalkaline phosphatase.

The cosmid DNA was then cleaved with the restriction enzyme BamHI(Amersham Pharmacia, Freiburg, Germany, Product Description BamHI, Codeno. 27-0868-04). The cosmid DNA treated in this manner was mixed withthe treated ATCC13032 DNA and the batch was treated with T4 DNA ligase(Amersham Pharmacia, Freiburg, Germany, Product DescriptionT4-DNA-Ligase, Code no.27-0870-04). The ligation mixture was then packedin phages with the aid of Gigapack II XL Packing Extract (Stratagene, LaJolla, USA, Product Description Gigapack II XL Packing Extract, Code no.200217).

For infection of the E. coli strain NM554 (Raleigh et al. 1988, NucleicAcid Res. 16:1563-1575) the cells were taken up in 10 mM MgSO₄ and mixedwith an aliquot of the phage suspension. The infection and titering ofthe cosmid library were carried out as described by Sambrook et al.(1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor), thecells being plated out on LB agar (Lennox, 1955, Virology, 1:190) +100μg/ml ampicillin. After incubation overnight at 37° C., recombinantindividual clones were selected.

Example 2

Isolation and Sequencing of the citA Gene

The cosmid DNA of an individual colony was isolated with the QiaprepSpin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) inaccordance with the manufacturer's instructions and partly cleaved withthe restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany,Product Description Sau3AI, Product No. 27-0913-02). The DNA fragmentswere dephosphorylated with shrimp alkaline phosphatase (Roche MolecularBiochemicals, Mannheim, Germany, Product Description SAP, Product No.1758250). After separation by gel electrophoresis, the cosmid fragmentsin the size range of 1500 to 2000 bp were isolated with the QiaExII GelExtraction Kit (Product No. 20021, Qiagen, Hilden, Germany).

The DNA of the sequencing vector pZero-1, obtained from Invitrogen(Groningen, The Netherlands, Product Description Zero Background CloningKit, Product No. K2500-01) was cleaved with the restriction enzyme BamHI(Amersham Pharmacia, Freiburg, Germany, Product Description BamHI,Product No. 27-0868-04). The ligation of the cosmid fragments in thesequencing vector pZero-1 was carried out as described by Sambrook etal. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor),the DNA mixture being incubated overnight with T4 ligase (PharmaciaBiotech, Freiburg, Germany). This ligation mixture was thenelectroporated (Tauch et al. 1994, FEMS Microbiol Letters, 123:343-7)into the E. coli strain DH5αMCR (Grant, 1990, Proceedings of theNational Academy of Sciences, U.S.A., 87:4645-4649) and plated out on LBagar (Lennox, 1955, Virology, 1:190) with 50 μg/ml zeocin.

The plasmid preparation of the recombinant clones was carried out withBiorobot 9600 (Product No. 900200, Qiagen, Hilden, Germany). Thesequencing was carried out by the dideoxy chain termination method ofSanger et al. (1977, Proceedings of the National Academies of Sciences,U.S.A., 74:5463-5467) with modifications according to Zimmermann et al.(1990, Nucleic Acids Research, 18:1067). The “RR dRhodamin TerminatorCycle Sequencing Kit” from PE Applied Biosystems (Product No. 403044,Weiterstadt, Germany) was used. The separation by gel electrophoresisand analysis of the sequencing reaction were carried out in a“Rotiphoresis NF Acrylamide/Bisacrylamide” Gel (29:1) (Product No.A124.1, Roth, Karlsruhe, Germany) with the “ABI Prism 377” sequencerfrom PE Applied Biosystems (Weiterstadt, Germany).

The raw sequence data obtained were then processed using the Stadenprogram package (1986, Nucleic Acids Research, 14:217-231) version 97-0.The individual sequences of the pZero1 derivatives were assembled to acontinuous contig. The computer-assisted coding region analysis [sic]were prepared with the XNIP program (Staden, 1986, Nucleic AcidsResearch, 14:217-231). Further analyses were carried out with the “BLASTsearch program” (Altschul et al., 1997, Nucleic Acids Research,25:3389-3402) against the non-redundant databank of the “National Centerfor Biotechnology Information” (NCBI, Bethesda, Md., USA).

The resulting nucleotide sequence is shown in SEQ ID No. 1. Analysis ofthe nucleotide sequence showed an open reading frame of 1653 bp, whichwas called the citA gene. The citA gene codes for a polypeptide of of[sic] 551 amino acids.

Example 3

Preparation of an Integration Vector for Integration Mutagenesis of thecitA Gene

From the strain ATCC 13032, chromosomal DNA was isolated by the methodof Eikmanns et al. (Microbiology 140: 1817-1828 (1994)). On the basis ofthe sequence of the citA gene known for C. glutamicum from example 2,the following oligonucleotides were chosen for the polymerase chainreaction (see SEQ ID No. 4 and SEQ ID No. 5): citA-int1: 5′ TTC CAG TCGGTG AGG TCA GT 3′ citA-int2: 5′ GTA CGA TCG CGG ATG GTT AC 3′

The primers shown were synthesized by MWG Biotech (Ebersberg, Germany)and the PCR reaction was carried out by the standard PCR method of Inniset al. (PCR protocols. A guide to methods and applications, 1990,Academic Press) with the Taq-polymerase from Boehringer Mannheim(Germany, Product Description Taq DNA polymerase, Product No. 1 146165). With the aid of the polymerase chain reaction, the primers allowamplification of an internal fragment of the citA gene 480 bp in size.The product amplified in this way was tested electrophoretically in a0.8% agarose gel.

The amplified DNA fragment (see SEQ ID No. 3) was ligated with the TOPOTA Cloning Kit from Invitrogen Corporation (Carlsbad, Calif., USA;Catalogue Number K4500-01) in the vector pCR2.1-TOPO (Mead at al. (1991)Bio/Technology 9:657-663).

The E. coli strain TOP10 was then electroporated with the ligation batch(Hanahan, In: DNA cloning. A practical approach. Vol. I. IRL-Press,Oxford, Washington DC, USA, 1985). Selection for plasmid-carrying cellswas made by plating out the transformation batch on LB agar (Sambrook etal., Molecular cloning: a laboratory manual. 2^(nd) Ed. Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), which had beensupplemented with 50 mg/l kanamycin. Plasmid DNA was isolated from atransformant with the aid of the QIAprep Spin Miniprep Kit from Qiagenand checked by restriction with the restriction enzyme EcoRI andsubsequent agarose gel electrophoresis (0.8%). The plasmid was calledpCR2.1 citAint and is shown in FIG. 1.

The following microorganism was deposited as a pure culture on23.01.2001 at the Deutsche Sammlung für Mikroorganismen und Zellkulturen(DSMZ=German Collection of Microorganisms and Cell Cultures,Braunschweig, Germany) in accordance with the Budapest Treaty:

-   Escherichia coli Top10/pCR2.1 citAint as DSM 13998

Example 4

Integration Mutagenesis of the citA Gene in the Strains DSM 5715 andFERM-BP 1763

The vector pCR2.1 citAint mentioned in example 3 was electroporated bythe electroporation method of Tauch et al.(FEMS Microbiological Letters,123:343-347 (1994)) into the strains Corynebacterium glutamicum DSM 5715and Brevibacterium lactofermentum FERM-BP 1763. The strain DSM 5715 isan AEC-resistant lysine producer (EP-B-0435132), and the strain FERM-BP1763 is a mycophenolic acid-resistant valine producer (U.S. Pat. No.5,188,948). The vector pCR2.1 citAint cannot replicate independently inDSM5715 and FERM-BP 1763 and is retained in the cell only if it hasintegrated into the chromosome of DSM 5715 or FERM-BP 1763. Selection ofclones with pCR2.1 citAint integrated into the chromosome was carriedout by plating out the electroporation batch on LB agar (Sambrook etal., Molecular cloning: a laboratory manual. 2^(nd) Ed. Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.), which had beensupplemented with 15 mg/l kanamycin.

For detection of the integration, the citAint fragment was labelled withthe Dig hybridization kit from Boehringer by the method of “The DIGSystem Users Guide for Filter Hybridization” of Boehringer Mannheim GmbH(Mannheim, Germany, 1993). Chromosomal DNA of in each case a potentialintegrant was isolated by the method of Eikmanns et al. (Microbiology140: 1817-1828 (1994)) and in each case cleaved with the restrictionenzymes EcoRI, BamHI and HindIII. The fragments formed were separated bymeans of agarose gel electrophoresis and hybridized at 68° C. with theDig hybrization [sic] kit from Boehringer. The plasmid pCR2.1 citAintmentioned in example 3 had been inserted into the chromosome of DSM5715and into the chromosome of FERM-BP 1763 within the chromosomal citAgene. The strains were called DSM5715::pCR2.1citAint and FERM-BP1763::pCR2.1citAint.

Example 5

Preparation of Lysine

The C. glutamicum strain DSM5715::pCR2.1citAint obtained in example 4was cultured in a nutrient medium suitable for the production of lysineand the lysine content in the culture supernatant was determined.

For this, the strain was first incubated on an agar plate with thecorresponding antibiotic (brain-heart agar with kanamycin (25 mg/l)[sic] for 24 hours at 33° C. Starting from this agar plate culture, apreculture was seeded (10 ml medium in a 100 ml conical flask). Thecomplete medium CgIII was used as the medium for the preculture. MediumCg III NaCl 2.5 g/l Bacto-Peptone 10 g/l Bacto-Yeast extract 10 g/lGlucose (autoclaved separately) 2% (w/v)The pH is brought to pH 7.4

Kanamycin (25 mg/l) was added to this. The preculture was incubated for16 hours at 33° C. at 240 rpm on a shaking machine. A main culture wasseeded from this preculture such that the initial OD (660 nm) of themain culture is 0.1 OD. Medium MM was used for the main culture. MediumMM CSL (corn steep liquor) 5 g/l MOPS (morpholinopropanesulfonic acid)20 g/l Glucose (autoclaved separately) 50 g/l Salts: (NH₄)₂SO₄) [sic] 25g/l KH₂PO₄ 0.1 g/l MgSO₄ * 7H₂O 1.0 g/l CaCl₂ * 2H₂O 10 mg/l FeSO₄ *7H₂O 10 mg/l MnSO₄ * H₂O 5.0 mg/l Biotin (sterile-filtered) 0.3 mg/lThiamine * HCl (sterile-filtered) 0.2 mg/l Leucine (sterile-filtered)0.1 g/l CaCO₃ 25 g/l

The CSL, MOPS and the salt solution were brought to pH 7 with aqueousammonia and autoclaved. The sterile substrate and vitamin solutions werethen added, and the CaCO₃ autoclaved in the dry state was added.

Culturing was carried out in a 10 ml volume in a 100 ml conical flaskwith baffles. Kanamycin (25 mg/l) was added. Culturing was carried outat 33° C. and 80% atmospheric humidity.

After 72 hours, the OD was determined at a measurement wavelength of 660nm with a Biomek 1000 (Beckmann Instruments GmbH, Munich). The amount oflysine formed was determined with an amino acid analyzer fromEppendorf-BioTronik (Hamburg, Germany) by ion exchange chromatographyand post-column derivatization with ninhydrin detection. The result ofthe experiment is shown in table 1. TABLE 1 Strain OD(660) Lysine HClg/l DSM5715 7.5 13.3 DSM5715::pCR2.1citAint 7.4 14.4

Example 6

Preparation of Valine

The B. lactofermentum strain FERM-BP 1763::pCR2.1citAint obtained inexample 4 was cultured in a nutrient medium suitable for the productionof valine and the valine content in the culture supernatant wasdetermined.

For this, the strain was first incubated on an agar plate with thecorresponding antibiotic (brain-heart agar with kanamycin (25 mg/l)[sic] for 24 hours at 33° C. Starting from this agar plate culture, apreculture was seeded (10 ml medium in a 100 ml conical flask). Thecomplete medium CgIII was used as the medium for the preculture.Kanamycin (25 mg/l) was added to this. The preculture was incubated for16 hours at 33° C. at 240 rpm on a shaking machine. A main culture wasseeded from this preculture such that the initial OD (660 nm) of themain culture was 0.1 OD. Medium MM was used for the main culture. MediumMM CSL (corn steep liquor) 5 g/l MOPS (morpholinopropanesulfonic acid)20 g/l Glucose (autoclaved separately) 50 g/l Salts: (NH₄)₂SO₄) [sic] 25g/l KH₂PO₄ 0.1 g/l MgSO₄ * 7H₂O 1.0 g/l CaCl₂ * 2H₂O 10 mg/l FeSO₄ *7H₂O 10 mg/l MnSO₄ * H₂O 5.0 mg/l Isoleucine (sterile-filtered) 0.1 g/lMethionine (sterile-filtered) 0.1 g/l Thiamine * HCl (sterile-filtered)0.2 mg/l Leucine (sterile-filtered) 0.1 g/l CaCO₃ 25 g/l

The CSL (corn steep liquor), MOPS (morpholinopropanesulfonic acid) andthe salt solution were brought to pH 7 with aqueous ammonia andautoclaved. The sterile substrate and vitamin solutions were then added,and the CaCO₃ autoclaved in the dry state was added.

Culturing was carried out in a 10 ml volume in a 100 ml conical flaskwith baffles. Kanamycin (25 mg/l) was added. Culturing was carried outat 33° C. and 80% atmospheric humidity.

After 48 hours, the OD was determined at a measurement wavelength of 660nm with a Biomek 1000 (Beckmann Instruments GmbH, Munich). The amount ofvaline formed was determined with an amino acid analyzer fromEppendorf-BioTronik (Hamburg, Germany) by ion exchange chromatographyand post-column derivatization with ninhydrin detection.

The result of the experiment is shown in table 2. TABLE 2 Strain OD(660)Valine HCl g/l FERM-BP 1763 12.1 7.5 FERM-BP 1763::pCR2.1citAint 13.510.8

1-19. (canceled)
 20. A process for producing L-amino acids, whichcomprises fermentation of coryneform bacterium which produce the desiredL-amino acid in which the intracellular activity of a polypeptideencoded by the citA gene comprising the amino acid sequence of SEQ IDNO: 2 is eliminated, wherein elimination is achieved by one or moremethods of mutagenesis selected from the group consisting of deletion,insertional mutagenesis due to homologous recombination, and transitionor transversion mutagenesis with incorporation of a non-sense mutationin the citA gene.
 21. The process according to claim 20, wherein thecitA gene of said coryneform bacterium comprises a polynucleotide havingthe nucleotide sequence of nucleotides 201 to 1853 of SEQ ID NO:1. 22.The process according to claim 20, wherein the citA gene of saidcoryneform bacterium comprises a polynucleotide having the nucleotidesequence shown in SEQ ID NO:1.
 23. The process according to claim 20,further comprising: a) concentrating the desired L-amino acid produced,in the medium or in the cells of the bacterium; and b) isolating theL-amino acid produced.
 24. The process according to claim 20, whereinthe L-amino acid produced is selected from the group consisting ofL-asparagine, L-threonine, L-serine, L-glutamic acid, L-glycine,L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucine,L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan, andL-arginine.
 25. The process according to claim 24, wherein the L-aminoacid produced is L-lysine.
 26. The process according to claim 20,wherein bacteria are employed in which one or more further gene or genesof the biosynthesis pathway of the desired L-amino acid is additionallyenhanced.
 27. The process according to claim 20, wherein one or morecoryneform bacterium genes selected from the group consisting of: (a)the dapA gene which codes for dihydrodipicolinate synthase, (b) the gapgene which codes for glycerolaldehyde 3-phosphate dehydrogenase, (c) thezwf gene which codes for glucose 6-phosphate dehydrogenase, (d) the pycgene which codes for pyruvate carboxylase, (e) the lysE gene which codesfor a protein of lysine export, (f) the lysC gene which codes for a feedback resistant aspartate kinase, (g) the zwal gene which codes for thezwal protein is overexpressed by increasing the copy number or placingsaid gene under a strong promoter during fermentation for the productionof the desired L-amino acid.
 28. The process according to claim 27,wherein the L-amino acid produced is L-lysine.
 29. The process accordingto claim 20, wherein bacteria are employed in which metabolic pathwayswhich reduce the formation of the desired L-amino acid are at leastpartly eliminated.
 30. The process according to claim 20, wherein theactivity (activities) of the polypeptide(s) encoded by one or morecoryneform bacterium genes selected from the group consisting of: (a)the pck gene which codes for pyhosphoenol pyruvate carbosykinase, (b)the pgi gene which codes for glucose 6-phosphate isomerase, (c) the poxBgene which codes for pyruvate oxidase (d) the zwa2 gene which codes forthe zwa2 protein is attenuated.
 31. The process according to claim 30,wherein the expression of one or more coryneform bacterium genesselected from the group consisting of: (a) the pck gene which codes forpyhosphoenol pyruvate carbosykinase, (b) the pgi gene which codes forglucose 6-phosphate isomerase, (c) the poxB gene which codes forpyruvate oxidase (d) the zwa2 gene which codes for the zwa2 protein isreduced or eliminated.
 32. The process according to claim 30, whereinthe catalytic properties of the polypeptide(s) encoded by one or morecoryneform bacterium genes selected from the group consisting of: (a)the pck gene which codes for pyhosphoenol pyruvate carbosykinase, (b)the pgi gene which codes for glucose 6-phosphate isomerase, (c) the poxBgene which codes for pyruvate oxidase (d) the zwa2 gene which codes forthe zwa2 protein are reduced or eliminated.
 33. The process according toclaim 20, wherein microorganisms of the genus Corynebacterium areemployed.
 34. The process according to claim 33, wherein microorganismsof the species Corynebacterium glutamicum are employed.